Methods and systems for cell and bead processing

ABSTRACT

The present disclosure provides methods and systems for cell and bead processing or analysis. A method for processing a cell or bead may comprise subjecting a bead to conditions sufficient to change a first characteristic or set of characteristics (e.g., cell or bead size). Such a method may further comprise subjecting the cell or bead to conditions sufficient to change a second characteristic or set of characteristics. In some cases, crosslinks may be formed within the cell or bead.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplications Nos. 62/689,651, filed Jun. 25, 2018, and 62/788,873, filedJan. 6, 2019, which applications are entirely incorporated herein byreference.

BACKGROUND

Samples may be processed for various purposes, such as identification ofa type of moiety within the sample. The sample may be a biologicalsample. The biological samples may be processed for various purposes,such as detection of a disease (e.g., cancer) or identification of aparticular species. There are various approaches for processing samples,such as polymerase chain reaction (PCR) and sequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Partitions and/or biological samples in partitions may be subjected tovarious processes, such as chemical processes or physical processes.Partitions and/or samples in partitions may be subjected to heating orcooling, or chemical reactions, such as to yield species that may bequalitatively or quantitatively processed.

SUMMARY

The present disclosure provides methods for use in various sampleprocessing and analysis applications. The methods provided herein mayincrease a concentration of a first set of molecules within a samplerelative to a second set of molecules within the same sample, therebyenriching the first set of molecules within the sample. Such methods maybe useful, for example, in controlled analysis and processing ofanalytes such as biological particles, nucleic acids, and proteins.

In an aspect, the present disclosure provides a method of processing acell, comprising: (a) subjecting the cell to conditions sufficient to:(i) change a cross-section of the cell from a first cross-section to asecond cross-section, which second cross-section is less than the firstcross-section, and (ii) form crosslinks within the cell having thesecond cross-section; and (b) providing the cell having the secondcross-section in an aqueous fluid.

In some embodiments, the crosslinks are formed upon cross-linking one ormore cross-linkable molecules within the cell. In some embodiments, theone or more cross-linkable molecules are one or more polymers.

In some embodiments, the crosslinks are formed upon polymerizing aplurality of monomers within the cell.

In some embodiments, the cross-section of the cell is changed from thefirst cross-section to the second cross-section concurrently withformation of the crosslinks within the cell.

In some embodiments, the crosslinks are formed subsequent to changingthe cross-section from the first cross-section to the secondcross-section.

In some embodiments, the second cross-section is substantiallymaintained in the aqueous fluid.

In some embodiments, the aqueous fluid is in a droplet as part of anemulsion.

In some embodiments, the volume of the droplet is less than 10,000 pL.In some embodiments, the volume of the droplet is less than 1,000 pL. Insome embodiments, the volume of the droplet is less than 500 pL. In someembodiments, the volume of the droplet is less than 100 pL. In someembodiments, the volume of the droplet is less than 50 pL. In someembodiments, the volume of the droplet is less than 10 pL.

In some embodiments, the aqueous fluid is in a well, such as a well of aplurality of wells.

In some embodiments, (a) comprises bringing the cell in contact with afirst chemical species and a second chemical species, wherein (i)comprises using the first chemical species to change the cross-sectionfrom the first-cross-section to the second cross-section, and wherein(ii) comprises using the second chemical species to form the crosslinkswithin the cell.

In some embodiments, the first or second chemical species is selectedfrom the group consisting of disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA),dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In someembodiments, the first and/or the second chemical species is selectedfrom the group consisting of an organic solvent or a cross-linkingagent. In some embodiments, the first chemical species is an organicsolvent and the second chemical species is a cross-linking agent. Insome embodiments, the organic solvent is acetone. In some embodiments,the organic solvent is an alcohol. In some embodiments, the alcohol ismethanol or ethanol. In some embodiments, the cross-linking agent isphotocleavable crosslinker. In some embodiments, the cross-linking agentis an aldehyde. In some embodiments, the cross-linking agent isformaldehyde or glutaraldehyde.

In some embodiments, the method further comprises providing the cell inan aqueous reaction mixture, wherein the cell comprises a targetmolecule, and performing one or more reactions using the targetmolecule. In some embodiments, the method further comprisesco-partitioning the cell in a partition and performing the one or morereactions in the partition. In some embodiments, the partition is adroplet. In some embodiments, the partition is a well. In someembodiments, the target molecule is a nucleic acid molecule and whereinthe partition further comprises a plurality of nucleic acid barcodemolecules, wherein each nucleic acid barcode molecule of the pluralityof nucleic acid barcode molecules comprises a sequence comprising acommon barcode sequence. In some embodiments, the plurality of nucleicacid barcode molecules are attached to a bead. In some embodiments,sequences of the plurality of nucleic acid barcode molecules arereleasably attached to the bead. In some embodiments, the method furthercomprises releasing the sequences of the plurality of nucleic acidbarcode molecules from the bead within the partition. In someembodiments, the bead is a gel bead. In some embodiments, the gel beadis degradable upon application of a stimulus. In some embodiments, thestimulus is a chemical stimulus. In some embodiments, the stimulus is areducing agent. In some embodiments, the partition further comprises thechemical stimulus. In some embodiments, the one or more reactionscomprise barcoding the target molecule using a nucleic acid barcodemolecule of the plurality of nucleic acid barcode molecules.

In some embodiments, the cross-section is a diameter of the cell.

In some embodiments, the cross-section is a volume of the cell.

In some embodiments, the second cross-section of the cell is reduced byat least 5% compared to the first cross-section. In some embodiments,the second cross-section of the cell is reduced by at least 10% comparedto the first cross-section. In some embodiments, the secondcross-section of the cell is reduced by at least 15% compared to thefirst cross-section. In some embodiments, the second cross-section ofthe cell is reduced by at least 25% compared to the first cross-section.In some embodiments, the second cross-section of the cell is reduced byat least 50% compared to the first cross-section.

In some embodiments, the change from the first cross-section of the cellto the second cross-section is irreversible.

In some embodiments, the change from the first cross-section of the cellto the second cross-section is reversible. In some embodiments, thechange from the first cross-section of the cell to the secondcross-section is reversible upon application of a stimulus. In someembodiments, the stimulus is selected from the group consisting of athermal stimulus, a photo stimulus, and a chemical stimulus. In someembodiments, the stimulus comprises a change in pH. In some embodiments,the stimulus comprises a reducing agent. In some embodiments, thereducing agent comprises dithiothreitol. In some embodiments, the methodfurther comprises applying the stimulus, wherein application of thestimulus reverses the change from the first cross-section to the secondcross-section by at least 75%.

In an aspect, the present disclosure provides a method of processing abead, comprising: subjecting the bead to conditions sufficient to changea cross-section of the bead from a first cross-section to a secondcross-section, which second cross-section is less than the firstcross-section.

In some embodiments, the bead is a cell bead. In some embodiments, thebead is a gel bead.

In some embodiments, the subjecting the bead to the conditions comprisesbringing the bead in contact with a chemical species, wherein thechemical species subjects the cross-section to change from thefirst-cross-section to the second cross-section.

In some embodiments, the chemical species is an organic solvent. In someembodiments, the organic solvent is acetone. In some embodiments, theorganic solvent is an alcohol.

In some embodiments, the subjecting the bead to the conditions compriseschanging the temperature. In some embodiments, the bead comprises apolymer that is responsive to temperature. In some embodiments, thepolymer is poly (N-isopropylacrylamide).

In some embodiments, the method further comprises partitioning the beadhaving the second cross-section into a partition. In some embodiments,the method further comprises co-partitioning into the partition the beadhaving the second cross-section and an additional bead. In someembodiments, the method further comprises subjecting the additional beadto additional conditions sufficient to change a cross-section of theadditional bead from a third cross-section to a fourth cross-section,which fourth cross-section is less than the third cross-section.

In some embodiments, the additional bead is a cell bead. In someembodiments, the additional bead is a gel bead.

In some embodiments, the additional conditions are the same as theconditions. In some embodiments, the additional conditions and theconditions are different. In some embodiments, the additional conditionsare applied prior to the co-partitioning. In some embodiments, theadditional conditions are applied during the co-partitioning. In someembodiments, the additional conditions are applied subsequent to theco-partitioning.

In some embodiments, the conditions are applied prior to thepartitioning. In some embodiments, the conditions are applied during thepartitioning. In some embodiments, the conditions are applied subsequentto the partitioning.

In some embodiments, the method further comprises co-partitioning intothe partition the bead having the second cross-section and a cell.

In some embodiments, the cell is subjected to additional conditionssufficient to: (i) change a cross-section of said cell from a firstcross-section to a second cross-section, which second cross-section isless than said first cross-section, and (ii) form crosslinks within saidcell having said second cross-section. In some embodiments, thepartition is a droplet (e.g., a droplet as part of an emulsion). In someembodiments, the partition is in a well (e.g., as part of a plurality ofwells).

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead.

FIG. 9 shows an example schematic of cell processing.

FIG. 10 shows an example architecture of a computer system programmed orotherwise configured to implement methods provided herein.

FIG. 11 illustrates an example scheme for generating an occupiedpartition having a reduced cross-section.

FIG. 12A illustrates solvent-mediated results of cross-section reductionof partitions and/or beads.

FIG. 12B illustrates results of in situ cross-section reduction ofbeads.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using an extension reaction, nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR,quantitative PCR, or real time PCR), or isothermal amplification. Suchsystems may provide a plurality of raw genetic data corresponding to thegenetic information of a subject (e.g., human), as generated by thesystems from a sample provided by the subject. In some examples, suchsystems provide sequencing reads (also “reads” herein). A read mayinclude a string of nucleic acid bases corresponding to a sequence of anucleic acid molecule that has been sequenced. In some situations,systems and methods provided herein may be used with proteomicinformation.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, macromolecules of biological particles suchas cellular macromolecules. The sample may be a cell sample. The samplemay be a cell line or cell culture sample. The sample can include one ormore cells. The sample can include one or more microbes. The biologicalsample may be a nucleic acid sample or protein sample. The biologicalsample may also be a carbohydrate sample or a lipid sample. Thebiological sample may be derived from another sample. The sample may bea tissue sample, such as a biopsy, core biopsy, needle aspirate, or fineneedle aspirate. The sample may be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample may be a skin sample.The sample may be a cheek swab. The sample may be a plasma or serumsample. The sample may be a cell-free or cell free sample. A cell-freesample may include extracellular polynucleotides. Extracellularpolynucleotides may be isolated from a bodily sample that may beselected from the group consisting of blood, plasma, serum, urine,saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent of a cell. The biologicalparticle may be or may include DNA, RNA, organelles, proteins, or anycombination thereof. The biological particle may be or may include amatrix (e.g., a gel or polymer matrix) comprising a cell or one or moreconstituents from a cell (e.g., cell bead), such as DNA, RNA,organelles, proteins, or any combination thereof, from the cell. Thebiological particle may be obtained from a tissue of a subject. Thebiological particle may be a hardened cell. Such hardened cell may ormay not include a cell wall or cell membrane. The biological particlemay include one or more constituents of a cell, but may not includeother constituents of the cell. An example of such constituents is anucleus or an organelle. A cell may be a live cell. The live cell may becapable of being cultured, for example, being cultured when enclosed ina gel or polymer matrix, or cultured when comprising a gel or polymermatrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA. The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

Provided herein are methods that may be used for various sampleprocessing and analysis applications. The methods provided herein maycomprise processing of a cell and/or a particle (e.g., bead, such as agel bead). The methods provided herein may alter one or morecharacteristics of the cell and/or particle, such as a size (e.g.,diameter or cross-section) and/or volume of the cell and/or particle.Methods comprising cell processing may provide cell beads comprising aplurality of crosslinks (e.g., a plurality of crosslinks between one ormore polymer molecules). A method of processing a cell may comprisesubjecting the cell to conditions sufficient to change a characteristic(e.g., a cross-section) of the cell. This process may involve the use ofa chemical species and may in some case result in polymerization withinand/or around the cell. Alternatively or in addition, the cell may becontacted with an additional chemical species, which additional chemicalspecies may further alter the characteristic of the cell or alter anadditional characteristic of the cell. The additional chemical speciesmay be used to promote the formation of crosslinks within the cell(e.g., to promote the formation of a polymer network within the cell). Acell may be processed within a bulk solution. Alternatively, a cell maybe processed within a partition such as a droplet or well. The droplet,well, or bulk solution may comprise an aqueous fluid. The cell and/orcomponent of the cell (e.g., nucleic acid molecules included therein)may undergo further processing, such as within a partition (e.g.,droplet or well). Particles may be subjected to similar processing. Forexample, a particle (e.g., bead) may be subjected to conditionssufficient to change a characteristic (e.g., cross-section) of theparticle. One or more chemical species may be used to change thecharacteristic of the particle. In some cases, a first chemical speciesmay be used to change the characteristic of the particle and a secondchemical species may be used to further alter the characteristic or tochange another characteristic of the particle. The present disclosurealso provides kits and compositions comprising cells and/or particlesprocessed according to the methods provided herein and kits forprocessing cells and/or particles according to the methods providedherein.

Methods of Processing Cells and Beads

In an aspect, the present disclosure provides a method for processing acell. The method may comprise subjecting a cell to conditions sufficientto change one or more characteristics of the cell and subsequentlyproviding the cell for further processing. In some cases, one or morephysical parameters or dimensions and/or one or more othercharacteristics of the cell may be changed. For example, a cross-sectionof the cell may be changed from a first cross-section to a secondcross-section. The first cross-section may be smaller or larger than thesecond cross-section. Alternatively or in addition, one or more othercharacteristics of the cell may be changed. For example, the fluidity,density, rigidity, porosity, or other characteristic of the cell or oneor more components thereof may be changed. In an example, a polymerand/or gel matrix may be formed within the cell. In another example,crosslinks (e.g., crosslinked proteins or polymers) may be formed withinthe cell. The same or different conditions may be used to change oraffect different characteristics of the cell at the same or differenttimes. In some cases, a first condition or set of conditions may be usedto change a first characteristic or set of characteristics of the cell(e.g., a cross-section) and a second condition or set of conditions maybe used to change a second characteristic or set of characteristics ofthe cell. The first condition or set of conditions may be applied at thesame or a different time as the second condition or set of conditions.For example, a first characteristic or set of characteristics may bechanged under a first condition or set of conditions, after which asecond characteristic or set of characteristics may be changed under asecond condition or set of conditions. The first condition or set ofconditions may comprise a first chemical species and the secondcondition or set of conditions may comprise a second chemical species,where the first chemical species and the second chemical species may bethe same or different. In some cases, the first chemical species may bean organic solvent and the second chemical species may be across-linking agent.

The cell may be derived from any suitable source. For example, the cellmay derive from a biological sample or an environmental sample. In somecases, the cell may derive from a bodily sample (e.g., as describedherein). For example, the cell may derive from a sample comprisingblood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool,tears and/or another bodily fluid. A sample comprising the cell may besubjected to various processing including, for example, filtration,centrifugation, extraction, washing, and elution. In some cases, thecell may be isolated from a biological sample or an environmentalsample. For example, the cell may be isolated from a blood sample.

The cell may be of any type (e.g., as described herein). For example,the cell may be a mammalian, fungal, plant, bacterial, or other celltype. In some cases, the cell is a mammalian cell, such as a human cell.The cell may be, for example, a stem cell, liver cell, nerve cell, bonecell, blood cell, reproductive cell, skin cell, skeletal muscle cell,cardiac muscle cell, smooth muscle cell, hair cell, hormone-secretingcell, or glandular cell. The cell may be, for example, an erythrocyte(e.g., red blood cell), a megakaryocyte (e.g., platelet precursor), amonocyte (e.g., white blood cell), a leukocyte, a B cell, a T cell (suchas a helper, suppressor, cytotoxic, or natural killer T cell), anosteoclast, a dendritic cell, a connective tissue macrophage, anepidermal Langerhans cell, a microglial cell, a granulocyte, a hybridomacell, a mast cell, a natural killer cell, a reticulocyte, ahematopoietic stem cell, a myoepithelial cell, a myeloid-derivedsuppressor cell, a platelet, a thymocyte, a satellite cell, anepithelial cell, an endothelial cell, an epididymal cell, a kidney cell,a liver cell, an adipocyte, a lipocyte, or a neuron cell. In some cases,the cell may be associated with a cancer, tumor, or neoplasm. In somecases, the cell may be associated with a fetus. In some cases, the cellmay be a Jurkat cell.

The cell may have any feature or dimension. For example, the cell mayhave a first dimension, a second dimension, and a third dimension, wherethe first, second, and third dimensions are approximately the same. Inother cases, the first and second dimensions may be approximately thesame, and the third dimension may be different, or the first, second,and third dimensions may all be different. In some cases, the cell maycomprise a dimension (e.g., a diameter) of at least about 1 μm. Forexample, the cell may comprise a dimension of at least about 1micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm,11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75μm, 80 μm, 85 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm,250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm,700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm), orgreater. In some cases, the cell may comprise a dimension of betweenabout 1 μm and 500 μm, such as between about 1 μm and 100 μm, betweenabout 100 μm and 200 μm, between about 200 μm and 300 μm, between about300 μm and 400 μm, or between about 400 μm and 500 μm. For example, thecell may comprise a dimension of between about 1 μm and 100 μm. Any orall dimensions of the cell may be variable. For example, the dimensionsof a substantially fluid cell may vary over a rapid timescale.Dimensions of a more rigid cell may be fixed or may vary with lesseramplitude. Accordingly, the dimensions provided herein may representaverages rather than fixed values. The volume of the cell may be atleast about 1 μm³. In some cases, the volume of the cell may be at leastabout 10 μm³. For example, the volume of the cell may be at least 1 μm³,2 μm³, 3 μm³, 4 μm³, 5 μm³, 6 μm³, 7 μm³, 8 μm³, 9 μm³, 10 μm³, 12 μm³,14 μm³, 16 μm³, 18 μm³, 20 μm³, 25 μm³, 30 μm³, 35 μm³, 40 μm³, 45 μm³,50 μm³, 55 μm³, 60 μm³, 65 μm³, 70 μm³, 75 μm³, 80 μm³, 85 μm³, 90 μm³,95 μm³, 100 μm³, 125 μm³, 150 μm³, 175 μm³, 200 μm³, 250 μm³, 300 μm³,350 μm³, 400 μm³, 450 μm³, μm³, 500 μm³, 550 μm³, 600 μm³, 650 μm³, 700μm³, 750 μm³, 800 μm³, 850 μm³, 900 μm³, 950 μm³, 1000 μm³, 1200 μm³,1400 μm³, 1600 μm³, 1800 μm³, 2000 μm³, 2200 μm³, 2400 μm³, 2600 μm³,2800 μm³, 3000 μm³, or greater. In some cases, the cell may comprise avolume of between about 1 μm³ and 100 μm³, such as between about 1 μm³and 10 μm³, between about 10 μm³ and 50 μm³, or between about 50 μm³ and100 μm³. In some cases, the cell may comprise a volume of between about100 μm³ and 1000 μm³, such as between about 100 μm³ and 500 μm³ orbetween about 500 μm³ and 1000 μm³. In some cases, the cell may comprisea volume between about 1000 μm³ and 3000 μm³, such as between about 1000μm³ and 2000 μm³ or between about 2000 μm³ and 3000 μm³. In some cases,the cell may comprise a volume between about 1 μm³ and 3000 μm³, such asbetween about 1 μm³ and 2000 μm³, between about 1 μm³ and 1000 μm³,between about 1 μm³ and 500 μm³, or between about 1 μm³ and 250 μm³.

The cell may comprise one or more cross-sections that may be the same ordifferent. In some cases, the cell may have a first cross-section thatis different from a second cross-section. The cell may have a firstcross-section that is at least about 1 μm. For example, the cell maycomprise a cross-section (e.g., a first cross-section) of at least about1 micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70μm, 75 μm, 80 μm, 85 μm, 90 μm, 100 μm, 120 μm, 140 μm, 160 μm, 180 μm,200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm,650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter(mm), or greater. In some cases, the cell may comprise a cross-section(e.g., a first cross-section) of between about 1 μm and 500 μm, such asbetween about 1 μm and 100 μm, between about 100 μm and 200 μm, betweenabout 200 μm and 300 μm, between about 300 μm and 400 μm, or betweenabout 400 μm and 500 μm. For example, the cell may comprise across-section (e.g., a first cross-section) of between about 1 μm and100 μm. In some cases, the cell may have a second cross-section that isat least about 1 μm. For example, the cell may comprise a secondcross-section of at least about 1 micrometer (um), 2μm, 3μm, 4μm, 5μm,6μm, 7μm, 8 μm, 9μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 100 μm, 120 μm, 140μm, 160 μm, 180 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950μm, 1 millimeter (mm), or greater. In some cases, the cell may comprisea second cross-section of between about 1 μm and 500 μm, such as betweenabout 1 μm and 100 μm, between about 100 μm and 200 um, between about200 μm and 300 μm, between about 300 μm and 400 μm, or between about 400um and 500 μm. For example, the cell may comprise a second cross-sectionof between about 1 μm and 100 μm.

A cross section (e.g., a first cross-section) may correspond to adiameter of the cell. In some cases, the cell may be approximatelyspherical. In such cases, the first cross-section may correspond to thediameter of the cell. In other cases, the cell may be approximatelycylindrical. In such cases, the first cross-section may correspond to adiameter, length, or width along the approximately cylindrical cell. Insome cases, the cell may comprise a surface. A cell surface may compriseone or more features. For example, a cell may comprise a dendriticreceiver, flagella, roughed border, or other feature.

A characteristic or set of characteristics of the cell may be changed byone or more conditions. A condition suitable for changing acharacteristic or set of characteristics of the cell may be, forexample, a temperature, a pH, an ion or salt concentration, a pressure,or another condition. For example, the cell may be exposed to a chemicalspecies that may bring about a change in one or more characteristics ofthe cell. In some cases, a stimulus may be used to change one or morecharacteristics of the cell. For example, upon application of thestimulus, one or more characteristics of the cell may be changed. Thestimulus may be, for example, a thermal stimulus, a photo stimulus, achemical stimulus, or another stimulus. In some cases, conditionssufficient to change the one or more characteristics of the cell maycomprise one or more different conditions, such as a temperature and apressure, a pH and a salt concentration, a chemical species and atemperature, or any other combination of conditions. A temperaturesufficient for changing one or more characteristics of the cell may be,for example, at least about 0 degrees Celsius (° C.), 1° C., 2° C., 3°C., 4° C., 5° C., 10° C., or higher. For example, the temperature may beabout 4° C. In other cases, a temperature sufficient for changing one ormore characteristics of the cell may be, for example, at least about 25°C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., or higher. Forexample, the temperature may be about 37° C. A pH sufficient forchanging one or more characteristics of the cell may be, for example,between about 5 and 8, such as between about 6 and 7.

In some cases, a chemical species or a chemical stimulus may be used tochange one or more characteristics of the cell. For example, a chemicalspecies or a chemical stimulus may be used to change a dimension of acell (e.g., a cross-section, diameter, or volume). In some cases, achemical species or a chemical stimulus may be used to change adimension of a cell (e.g., a cross-sectional diameter) from a firstdimension to a second dimension (e.g., a second cross-sectionaldimeter), where the second dimension is reduced compared to the firstdimension. The chemical species may comprise an organic solvent, such asan alcohol, ketone, or aldehyde. For example, the chemical species maycomprise acetone, methanol, ethanol, formaldehyde, or glutaraldehyde.The chemical species may comprise a cross-linking agent. For example,the chemical species may be selected from the group consisting ofdisuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, anddimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP),disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidylsuccinate) (EGS). In some cases, a cross-linking agent may be aphotocleavable cross-linking agent. In some cases, a chemical stimulusmay be used to change one or more characteristics of the cell (e.g., adimension of a cell), where the chemical stimulus comprises one or morechemical species. For example, the chemical stimulus may comprise afirst chemical species and a second chemical species, where the firstchemical species is an organic solvent and the second chemical speciesis a cross-linking agent. In some cases, a chemical stimulus maycomprise a chemical species that is a fixation agent that is capable offixing or preserving a cell. For example, an organic solvent such as analcohol (e.g., ethanol or methanol), ketone (e.g., acetone), or aldehyde(e.g., formaldehyde or glutaraldehyde) may act as a fixation agent.Alternatively, or in addition, a cross-linking agent may act as afixation agent. In some cases, a fixation agent may be selected from thegroup consisting of disuccinimidyl suberate (DSS), dimethylsuberimidate(DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidylpropionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycolbis(succinimidyl succinate) (EGS). In some cases, a first chemicalspecies and/or fixation agent may be provided to or brought into contactwith the cell to bring about a change in a first characteristic or setof characteristics of the cell, and a second chemical species and/orfixation agent may be provided to or brought into contact with the cellto bring about a change in a second characteristic or set ofcharacteristics of the cell. For example, a first chemical speciesand/or fixation agent may be provided to or brought into contact withthe cell to bring about a change in a dimension of a cell (e.g., areduction in cross-sectional diameter), and a second chemical speciesand/or fixation agent may be provided to or brought into contact withthe cell to bring about a change in a second characteristic or set ofcharacteristics of the cell (e.g., forming crosslinks within and/orsurrounding the cell). The first and second chemical species and/orfixation agents may be provided to or brought into contact with the cellat the same or different times.

Fixation may comprise dehydration of the cell. In some cases, fixationmay affect one or more parameters or characteristics of the cell. Forexample, fixation may result in shrinkage or size reduction of the cell.Providing a fixation agent to the cell may result in a change in adimension of the cell. For example, providing a fixation agent to thecell may result in a change in the volume or diameter of the cell.Providing a fixation agent to the cell may result in a change in across-section of the cell (e.g., a cross-sectional diameter). Forexample, a first cross-section of the cell prior to fixation may bedifferent (e.g., larger) than a second cross-section of the cellfollowing fixation. In an example, an approximately spherical cell maycomprise a first cross section (e.g., a cross-sectional diameter) priorto fixation that is reduced in size to a second cross-section followingfixation. Providing a fixation agent to the cell may result in a secondcross-section that is reduced by at least about 5% compared to the firstcross-section. In some cases, the second cross-section may be reduced byat least 6%, 8%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, or morerelative to the first cross-section. For example, the secondcross-section may be reduced by at least about 10%, 15%, 25%, or 50%relative to the first cross-section. Fixation may also affect otherfeatures of the cell. For example, fixation may result in a change inthe porosity of a membrane or wall of a cell; reorganization ofcomponents of the cell; a change in cell fluidity or rigidity; or otherchanges. In an example, a first fixation agent that is an organicsolvent is provided to the cell to change a first characteristic (e.g.,cell size) and a second fixation agent that is a cross-linking agent isprovided to the cell to change a second characteristic (e.g., cellfluidity or rigidity). The first fixation agent may be provided to thecell before the second fixation agent.

In some instances, an approximately spherical cell may comprise a firstdiameter prior to being subjected to a first set of conditions (e.g.,fixation, such as fixation achieved using an organic solvent) that isreduced in size compared to a second diameter following such processing(e.g., fixation) when maintained in a non-aqueous environment. Followingthis reduction in size to the second diameter, when maintained in anaqueous environment, the cell may increase in size to have a diametersubstantially similar to the first diameter. In some cases, anapproximately spherical cell may comprise a first diameter prior tobeing subjected to the first set of conditions (e.g., fixation, such asfixation achieved using an organic solvent) that is reduced in sizecompared to a second diameter following subjecting the cell to the firstset of conditions. Following this reduction in size to the seconddiameter, the cell may be cross-linked by a second chemical species(e.g., second fixative), wherein the second diameter is substantiallymaintained in an aqueous environment following cross-linking by thesecond chemical species (e.g., second fixative).

A change to a characteristic or set of characteristics of the cell maybe reversible or irreversible. In some cases, a change to acharacteristic or set of characteristics of the cell may besubstantially irreversible, such that the change cannot be readilyundone. For example, the size, morphology, or other feature of the cellmay be altered in a way that cannot be readily reversed. In an example,the change from a first cross-section of the cell to a secondcross-section of the cell is irreversible. In some cases, anirreversible change may be at least partially reversed upon theapplication of appropriate conditions and/or over a period of time. Inother cases, a change to a characteristic or set of characteristics ofthe cell may be at least partially reversible. For example, the size ofa cell may be reduced upon being subjected to a first condition or setof conditions (e.g., fixation using a first chemical species, such as anorganic solvent), and the size of a cell may be increased toapproximately the original size upon being subjected to a secondcondition or set of conditions (e.g., rehydration within an aqueousenvironment). Thus, the change from a first cross-section of the cell tothe second cross-section may be reversible. A reversible change (e.g., areversible size reduction) may be useful in, for example, providing acell of a given size to a given location, such as a partition. In somecases, a change to a characteristic or set of characteristics of thecell may be only partially reversible. For example, the size of a cellmay be reduced (e.g., by dehydration), and the reduction in cell sizemay be accompanied by reorganization of components within the cell. Uponreversal of the size of the cell (e.g., by rehydration), the arrangementof one or more cellular components may not revert to the originalarrangement of the cell prior to the size reduction. In another example,the size of the cell may be reduced. Upon reversal of the size of thecell, the size of the cell may not revert to the original size of thecell prior to the size reduction. A change to a characteristic or set ofcharacteristics may be reversible by at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or more, wherein the percentage is determined by thepercent difference in the characteristics or set of characteristicsafter reversal compared to the characteristics or set of characteristicsprior to being subjected to a first condition and a 100% reversibilityindicates a complete reversal to the original characteristics or set ofcharacteristics of the cell. A change to a characteristic or set ofcharacteristics of the cell, such as a cross-section of the cell, may bereversible upon application of a stimulus. The stimulus may be, forexample, a thermal stimulus, a photo stimulus, or a chemical stimulus.In some cases, the stimulus may comprise a change in pH and/orapplication of a reducing agent such as dithiothreitol. Application ofthe stimulus may reverse, wholly or in part, a change from, for example,a first cross-section to a second cross-section.

In some cases, a polymer or gel matrix may be formed within the cell.The formation of a polymer or gel matrix within the cell may result in achange in one or more characteristics of the cell, such as the size,fluidity, porosity, rigidity, organization, or one or more otherfeatures of the cell. A polymer or gel matrix may be formed, forexample, upon cross-linking one or more cross-linkable molecules withinthe cell. For example, a polymer or gel matrix may be formed uponcross-linking one or more molecules within the cell. The polymer or gelmatrix may be formed upon polymerizing a plurality of monomers withinthe cell. For examples, the plurality of monomers may comprise aminoacids or nucleic acids. The polymer or gel matrix may be formed uponpolymerizing a plurality of polymers within the cell. For example, thepolymers may comprise proteins, DNA, or RNA. Polymeric or gel precursorsmay be provided to the cell and may not form a polymer or gel matrixwithout application of a stimulus (e.g., as described herein). In somecases, the cell and/or components thereof may be encapsulated (e.g.,entrained or entrapped) within the polymer or gel matrix. Formation of apolymer or gel matrix within the cell may take place following one ormore other changes to the cell that may be brought about by one or moreother conditions.

For example, a first condition or set of conditions may be used tochange a first characteristic or set of characteristics of the cell anda polymer or gel matrix may be formed within the cell. The firstcondition or set of conditions may be used to change a cross-section ofthe cell from a first cross-section to a second cross-section (e.g., asdescribed herein). In some cases, the cross-section may be changed froma first cross-section to a second cross-section and the polymer or gelmatrix may be formed concurrently. The conditions used to bring aboutthese changes may be the same, partially the same, or different. Forexample, a first condition may be used to bring about the change in thesize of the cell and a second condition such as a stimulus (e.g., asdescribed herein) may be used to cause the polymer or gel matrix to formwithin the cell. Alternatively, a first condition or set of conditionsmay be used to reduce the size of the cell and cause the polymer or gelmatrix to form at the same time. For example, the same chemical speciesor collection of chemical species may be used to both change the size ofthe cell and form the polymer or gel matrix.

In some cases, the cross-section may be changed from a firstcross-section to a second cross-section and the polymer or gel matrixmay be subsequently formed. The conditions used to bring about thesechanges may be different. For example, a first chemical species orchemical stimulus, optionally in combination with one or more additionalconditions, may be used to change the size of the cell and a secondchemical species or chemical stimulus, optionally in combination withone or more additional conditions, may be used to form the polymer orgel matrix within the cell.

In some cases, the polymer or gel matrix may be formed, and the crosssection of the polymer or gel matrix may be subsequently changed. Forexample, a first chemical species or stimulus, optionally in combinationwith one or more additional conditions, may be used to form the polymeror gel matrix, followed by a second chemical species or chemicalstimulus, optionally in combination with one or more additionalconditions, may be used to change the size of the cell. In some cases,the cell may be lysed when subjected to a chemical species or stimulus,optionally in combination with one or more additional conditions, usedto form the polymer or gel matrix. For example, the formation of thepolymer network or gel may cause sufficient force on the cell causing itto lyse. In some cases, the cell may be lysed when subjected to achemical species or stimulus, optionally in combination with one or moreadditional conditions, used to change the cross-section from a firstcross-section to a second cross-section of the polymer or gel matrix. Insome cases, the second cross-section may be reduced by at least 6%, 8%,10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%,85%, 90%, or more relative to the first cross-section. For example, thesecond cross-section may be reduced by at least about 10%, 15%, 25%, or50% relative to the first cross-section. In some cases, the cell may belysed only when subjected to both a first chemical species and a secondchemical species used for forming the polymer or gel matrix and forchanging the cell cross-section, respectively.

A cell comprising a polymer or gel matrix may be referred to as a cellbead. A cell bead may be prepared by any useful method (e.g., asdescribed herein). Similarly, a particle (e.g., a bead, such as a gelbead) used to process a cell or cell bead or one or more components(e.g., nucleic acid molecules) thereof may be prepared by any usefulmethod. In some cases, preparing a cell bead may comprise combining acell, such as a cell that has been subjected to conditions to change oneor more characteristics of the cell, and one or more polymers and/orpolymer precursors and subjecting the combination to a polymerizationcondition. In an example, the one or more polymers and/or polymerprecursors may comprise two different polymers and/or polymer precursorsand generating the cell bead comprises subjecting the combination to acondition sufficient to generate a plurality of linkages between the twodifferent polymers and/or polymer precursors. The plurality of linkagesmay comprise triazole moieties. In such a system, click chemistry may beused to generate the linkages between the two different polymers and/orpolymer precursors. For example, the first polymer or polymer precursormay comprise a plurality of azide moieties and the second polymer orpolymer precursor may comprise a plurality of alkyne moieties. Reactionbetween azide and alkyne moieties may be promoted via a copper catalystor the use of strained alkynes, for example. A particle (e.g., bead)used to process components of a cell or cell bead (e.g., a beadcomprising a nucleic acid barcode molecule) may be similarly generated,where one or more polymers or polymer precursors may be linked to thenucleic acid barcode molecule prior to generation of the bead.Alternatively, the nucleic acid barcode molecule may be attached to thebead subsequent to its generation.

Similarly, a bead, a polymer, or polymer precursors (e.g., monomer unitsthat may polymerize and form said polymer) may be coupled to, attachedto, functionalized, or otherwise associated with a molecule (e.g., anucleic acid molecule or a nucleic acid barcode molecule) usingconjugation chemistry. Conjugation chemistry as described herein refersto any suitable chemical reaction that links, couples, or attaches afirst molecule (e.g., a polymer or a bead) with a second molecule (e.g.,a binding agent such as an analyte-specific binding agent, a nucleicacid molecule, a nucleic acid barcode molecule, or any other molecule).Conjugation chemistry may comprise bioconjugation chemistry and clickchemistry. Conjugation chemistry may comprise biological interactions(e.g., biotin/strepdavidin interactions) and/or bioorthogonal reactions.In some cases, coupling or attachment of molecules (e.g., nucleic acids,binding agents, etc.) to a bead, cell bead, or polymer as describedherein may be performed using click chemistry.

Click chemistry, as described herein, may comprise any type of clickreaction suitable for the functionalization of beads, cell beads,polymers, or precursors thereof with various types of molecules such asnucleic acid molecules, nucleic acid barcode molecules, binding agents(e.g., analyte-specific binding agents), etc. Examples of clickchemistry reactions (also referred to herein as “click reactions”) thatmay be used in combination with the methods, systems, and kits providedherein include, but are not limited to, transition-metal catalyzed orstrain-promoted azide-alkyne cycloadditions (e.g., Huisgen azide-alkyne1,3-dipolar cycloaddition, copper-catalyzed azide-alkyne cycloaddition(CuAAC), strain-promoted alkyne-azide cycloaddition, and/orruthenium-catalyzed azide-alkyne cycloaddition (RuAAC)), Diels-Alderreactions such as inverse-electron demand Diels-Alder reaction (e.g.,tetrazine-trans-cyclooctene reactions), or photo-click reactions (e.g.,alkene-tetrazole photoreactions).

A bead, cell bead, polymer, or precursor thereof may be attached to oneor more sets of molecules (e.g., binding agents, nucleic acid molecules,and/or nucleic acid barcode molecules) using such click chemistry. Thus,a bead, cell bead, polymer, or precursor thereof may comprise (e.g., maybe functionalized with) a first functional group. The first functionalgroup may be a first reactant for a click reaction. The one or more setsof molecules (e.g., binding agents and/or nucleic acid molecules) thatmay be attached to the bead, cell bead, polymer, or precursor thereof,may comprise a second functional group. The second functional group maybe a second reactant for a click reaction. The click reaction may be acopper-catalyzed azide-alkyne cycloaddition reaction, aninverse-electron demand Diels-Alder reaction, an avidin-biotininteraction, etc. In an example, a bead, cell bead, polymer, orprecursor thereof is modified with an analyte-specific binding agentusing a copper-catalyzed azide-alkyne cycloaddition click reaction. Suchreaction can comprise an azide-functionalized analyte-specific bindingagent and an alkyne-functionalized bead, cell bead, polymer, orprecursor thereof (e.g., a cell bead comprising a cell encapsulated in apolymer matric (e.g., a hydrogel matrix)). Click reaction may occurbetween the azide-functionalized analyte-specific binding agent andalkyne-functionalized bead, cell bead, polymer, or precursor thereof,thereby attaching the binding agent to the bead, cell bead, polymer, orprecursor thereof.

In addition to using click chemistry, a molecule (e.g., a binding agentor nucleic acid molecule) can be attached to a bead, cell bead, polymer,or precursor thereof, using various other bioconjugation or couplingmethods. Such bioconjugation methods can include various conjugationstrategies and functional group modifications such as mesylateformation, sulfur alkylation, NHS ester formation, carbamate formation,carbonate formation, amide bond formation, or any combination thereof.Such strategies and functional group modifications can be used forvarious reaction types such nucleophilic and/or electrophilicsubstitution reaction, nucleophilic and/or electrophilic additionreaction, and other suitable reaction types. In some cases, activatedcarboxylic acids can react with nucleophiles such as amines. In somecases, the carboxylic acid can be attached to a bead, cell bead,polymer, or precursor thereof, and the nucleophilic group such as anamine can be attached to the molecule (e.g., a nucleic acid molecule ora binding agent) to be attached to said bead, cell bead, polymer, orprecursor thereof. Such amide bond formation reactions can includeEDC/NHS (e.g., via 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC)and N-hydroxysuccinimide (NETS) or4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) mediated coupling reactions, wherein an activated ester (e.g.,an NHS ester attached to a bead surface) can react with an amine (e.g.,an amine of a nucleic acid molecule) to form an amide bond, therebyattaching said molecule (e.g., a nucleic acid molecule) to said bead(e.g., a gel bead). Any other suitable bioconjugation reactions can beused to attach a molecule to a bead.

Subsequent to being subjected to conditions sufficient to change one ormore characteristics of a cell (e.g., as described herein), the cell maybe provided in an aqueous fluid. In some cases, the cell may be includedwithin an aqueous fluid (e.g., in a solution or emulsion) prior to beingsubjected to such conditions. In some cases, the cell may undergo one ormore changes upon being subjected to such conditions and then undergoone or more further processes such as filtration, extraction, washing,elution, centrifugation, agitation, or isolation. Subsequent to any suchprocessing, the cell may be provided in the aqueous fluid. The aqueousfluid comprising the cell may be contained within a vessel or reservoir.In some cases, the aqueous fluid in which the processed cell is providedmay be stored for at least 24 hours, 48 hours, 72 hours, 120 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,12 months, or longer. In some cases, one or more stabilizing orpreserving agents may be included within the aqueous fluid. Stabilizingand preserving agents may be used to preserve the morphology, size, andother features of the cell. In some cases, the aqueous fluid may becooled or frozen. In some cases, the aqueous fluid may be used toprovide the cell for further processing and analysis. For example, oneor more reagents useful for processing or analyzing the cell orcomponents thereof may be added to the aqueous fluid. Reagents usefulfor processing or analyzing the cell may be selected from the groupconsisting of lysis agents or buffers, permeabilizing agents, enzymes(e.g., enzymes capable of digesting one or more RNA molecules, extendingone or more nucleic acid molecules, reverse transcribing an RNAmolecule, permeabilizing or lysing a cell, or carrying out otheractions, such as nucleases, restriction enzymes, transposases,polymerases, ligases, exonucleases, reverse transcriptases, and otherenzymes), fluorophores, oligonucleotides, primers, barcodes, nucleicacid barcode molecules (e.g., nucleic acid barcode molecules comprisingone or more barcode sequences), buffers, deoxynucleotide triphosphates,detergents, reducing agents, chelating agents, oxidizing agents,nanoparticles, beads, and antibodies. In some cases, one or moretemperature-sensitive enzymes, pH-sensitive enzymes, light-sensitiveenzymes, reverse transcriptases, proteases, ligase, polymerases,restriction enzymes, nucleases, protease inhibitors, exonucleases,and/or nuclease inhibitors may be added to the aqueous fluid. In somecases, the aqueous fluid comprising the cell may be transferred toand/or flowed through a channel of a microfluidic device. The aqueousfluid comprising the cell may be flowed through a junction of two ormore channels or expelled through a nozzle to generate a dropletcomprising the cell (e.g., as described herein).

In some cases, the aqueous fluid is in a droplet. For example, theaqueous fluid may be in a droplet as part of an emulsion, such as awater-in-oil emulsion. The volume of the droplet may be less than about100,000 pL. For example, the volume of the droplet may be less thanabout 10,000 pL, 1,000 pL, 500 pL, 100 pL, 50 pL, or 10 pL. In somecases, the aqueous fluid is a well as part of a plurality of wells. Eachwell of the plurality of wells may comprise one or more cells (e.g., asdescribed herein). The volume of the well may be less than about 100,000pL. For example, the volume of the well may be less than about 10,000pL, 1,000 pL, 500 pL, 100 pL, 50 pL, or 10 pL.

The aqueous fluid comprising the cell may comprise a target molecule ofinterest. The target molecule may be a nucleic acid molecule that maycomprise a sequence of interest. The target nucleic acid molecule maybe, for example, a deoxyribonucleic acid (DNA) molecule or a ribonucleicacid (RNA) molecule. Examples of RNA include, but are not limited to,messenger RNA (mRNA), ribosomal RNA (rRNA), mitochondrial RNA (mtRNA),small nucleolar RNA (snoRBA) and transfer RNA (tRNA). The RNA may be atranscript. The RNA may be small RNA that are less than 200 nucleic acidbases in length, or large RNA that are greater than 200 nucleic acidbases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5SrRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA(siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA),tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). TheRNA may be double-stranded RNA or single-stranded RNA. The RNA may becircular RNA. Similarly, DNA may be double-stranded DNA orsingle-stranded DNA. The DNA may comprise chromatin. A nucleic acidmolecule of interest may have any useful length, base composition, andother characteristics. In some cases, the aqueous fluid comprises anaqueous reaction mixture and the cell comprises a target molecule, andthe method comprises performing one or more reactions using the targetmolecule. In some cases, one or more such reactions are performed withina partition (e.g., as described herein).

In some cases, a target molecule of interest (e.g., a nucleic acidmolecule) may be included within the cell. For example, the targetmolecule of interest may be included in a nucleus of a cell. Access to atarget molecule included in the cell may be provided by lysing orpermeabilizing the cell. Lysing the cell may release the target moleculecontained therein from a cell. The cell may be lysed using a lysis agentsuch as a bioactive agent. A bioactive agent useful for lysing the cellmay be, for example, an enzyme (e.g., as described herein). An enzymeused to lyse the cell may or may not be capable of carrying outadditional actions such as degrading one or more RNA molecules.Alternatively, an ionic, zwitterionic, or non-ionic surfactant may beused to lyse the cell. Examples of surfactants include, but are notlimited to, TritonX-100, Tween 20, sarcosyl, or sodium dodecyl sulfate.Cell lysis may also be achieved using a cellular disruption method suchas an electroporation or a thermal, acoustic, or mechanical disruptionmethod. Alternatively, the cell may be permeabilized to provide accessto a target molecule included therein. Permeabilization may involvepartially or completely dissolving or disrupting a cell membrane or aportion thereof. Permeabilization may be achieved by, for example,contacting a cell membrane with an organic solvent or a detergent suchas Triton X-100 or NP-40.

The cell may be partitioned within a partition such as a well ordroplet, e.g., as described herein. For example, the cell providedwithin the aqueous fluid may be partitioned using a fluid that isimmiscible with the aqueous fluid. One or more reagents may beco-partitioned with the cell. For example, the cell may beco-partitioned with one or more reagents selected from the groupconsisting of lysis agents or buffers, permeabilizing agents, enzymes(e.g., enzymes capable of digesting one or more RNA molecules, extendingone or more nucleic acid molecules, reverse transcribing an RNAmolecule, permeabilizing or lysing a cell, or carrying out otheractions), fluorophores, oligonucleotides, primers, barcodes, nucleicacid barcode molecules (e.g., nucleic acid barcode molecules comprisingone or more barcode sequences), buffers, deoxynucleotide triphosphates,detergents, reducing agents, chelating agents, oxidizing agents,nanoparticles, beads, and antibodies. In some cases, the cell may beco-partitioned with one or more reagents selected from the groupconsisting of temperature-sensitive enzymes, pH-sensitive enzymes,light-sensitive enzymes, reverse transcriptases, proteases, ligase,polymerases, restriction enzymes, nucleases, protease inhibitors,exonucleases, transposases, and nuclease inhibitors. Similarly, the cellmay include or be co-partitioned with one or more target molecules. Insome cases, the partition comprises an aqueous reaction mixture and thecell comprises a target molecule, and the method comprises performingone or more reactions using the target molecule. In an example, thepartition comprises an aqueous reaction mixture comprising a pluralityof nucleic acid barcode molecules comprising a common barcode sequence.Partitioning the cell and one or more reagents may comprise flowing afirst phase comprising an aqueous fluid, the cell, and the one or morereagents and a second phase comprising a fluid that is immiscible withthe aqueous fluid toward a junction. Upon interaction of the first andsecond phases, a discrete droplet of the first phase comprising the celland the one or more reagents may be formed. In some cases, the partitionmay comprise a single cell. The cell may be lysed or permeabilizedwithin the partition (e.g., droplet) to provide access to a targetmolecule of interest within the cell. Accordingly, molecules originatingfrom the same cell may be isolated within the same partition.

A partition (e.g., droplet in emulsion) comprising a cell, or gel matrixderived thereof, that has been subjected to one or more conditions orsets of conditions described herein may have a first cross-section thatis smaller than a second cross-section of a partition comprising a cell,or gel matrix derived thereof, that has not been subjected to one ormore conditions or sets of conditions described herein. In some cases,the first cross-section may be smaller by at least 6%, 8%, 10%, 15%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% ,70%, 75%, 80%, 85%, 90%, ormore relative to the second cross-section. For example, the firstcross-section may be smaller by at least about 10%, 15%, 25%, or 50%relative to the second cross-section.

In some cases, the methods of the present disclosure may be performedwithin a partition. For example, the cell may be provided within apartition (e.g., a droplet or well) for processing. The cell may besubjected to conditions sufficient to change one or more characteristicsof the cell (e.g., as described herein). For example, a cross-section ofthe cell may be changed from a first cross-section to a secondcross-section within the partition. Similarly, a polymer or gel matrixmay be formed within the cell within the partition.

A partition (e.g., a well or droplet) comprising the cell may furthercomprise a bead (e.g., as described herein). In some instances, a cellmay be introduced to a partition comprising a bead (e.g., a gel bead).

In some cases, a bead may be subjected to processing as describedelsewhere herein with regard to cells. For example, the bead may besubjected to processing prior to partitioning into a partition.Alternatively or in addition, the bead may be subjected to processingduring or subsequent to partitioning in the partition. In some cases,one or more physical parameters or dimensions (e.g., volume anddiameter) and/or one or more other characteristics of the bead may bechanged. For example, a cross-section of the bead may be changed from afirst cross-section to a second cross-section. The first cross-sectionmay be smaller or larger than the second cross-section. In some cases,the second cross-section may be reduced by at least 6%, 8%, 10%, 15%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, ormore relative to the first cross-section. For example, the secondcross-section may be reduced by at least about 10%, 15%, 25%, or 50%relative to the first cross-section. Alternatively or in addition, oneor more other characteristics of the bead may be changed. For example,the fluidity, density, rigidity, porosity, refractive index, polarity,or other characteristic of the bead or one or more components thereofmay be changed.

A characteristic or set of characteristics of the bead may be changed byone or more conditions. A condition suitable for changing acharacteristic or set of characteristics of the bead may be, forexample, a temperature, a pH, an ion or salt concentration, a pressure,or another condition. For example, the bead may be exposed to a chemicalspecies that may bring about a change in one or more characteristics ofthe bead. In some cases, a stimulus may be used to change one or morecharacteristics of the bead. For example, upon application of thestimulus, one or more characteristics of the bead may be changed. Thestimulus may be, for example, a thermal stimulus, a photo stimulus, achemical stimulus, or another stimulus. In some cases, conditionssufficient to change the one or more characteristics of the bead maycomprise one or more different conditions, such as a temperature and apressure, a pH and a salt concentration, a chemical species and atemperature, or any other combination of conditions. A pH sufficient forchanging one or more characteristics of the bead may be, for example,between about 5 and 8, such as between about 6 and 7.

In some cases, a temperature change may be used to change thecharacteristics of the bead. The bead may comprise a temperatureresponsive polymer such as Poly-N (isopropylacrylamide). For example,changing the temperature may result in changing a dimension of the bead.A temperature sufficient for changing one or more characteristics of thecell may be, for example, at least about 0 degrees Celsius (° C.), 1°C., 2° C., 3° C., 4° C., 5° C., 10° C., or higher. For example, thetemperature may be about 4° C. In other cases, a temperature sufficientfor changing one or more characteristics of the cell may be, forexample, at least about 25° C., 30° C., 35° C., 37° C., 40° C., 45° C.,50° C., or higher. For example, the temperature may be about 37° C.

In some cases, a change to one or more characteristics of the bead maybe mediated by a chemical species or a chemical stimulus. For example, achemical species or chemical stimulus may be used to change a dimensionof the bead (e.g., a cross-section, diameter, or volume). The chemicalspecies may be one of those used for processing cells as describedelsewhere herein. In some cases, the chemical species is a fixationagent as described elsewhere herein.

A partition (e.g., droplet in emulsion) comprising a bead that has beensubjected to one or more conditions or sets of conditions describedherein may have a first cross-section that is smaller than a secondcross-section of a partition comprising a bead that has not beensubjected to one or more conditions or sets of conditions describedherein. In some cases, the first cross-section may be smaller by atleast 6%, 8%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or more relative to the second cross-section.For example, the first cross-section may be smaller by at least about10%, 15%, 25%, or 50% relative to the second cross-section.

A first partition (e.g., droplet in emulsion) comprising both a cell(e.g., a cell processed as described herein, such as a cell beadcomprising a polymer or gel matrix) and a bead that has been processedto change one or more of its characteristics (e.g., as described herein)may have a first cross-section that is smaller than a secondcross-section of a second partition comprising both a cell (e.g., a cellprocessed as described herein, such as a cell bead comprising a polymeror gel matrix) and a bead that has not been process to change one ormore of its characteristics. In some cases, the first cross-section maybe smaller by at least 6%, 8%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more relative to the secondcross-section. For example, the first cross-section may be smaller by atleast about 10%, 15%, 25%, or 50% relative to the second cross-section.

FIG. 11 illustrates an example scheme for generating an occupiedpartition having a reduced cross-section. In a first operation a firstparticle 1102A and a second particle 1104A may be provided. For example,the first particle 1102A may be a cell or a cell bead comprising apolymer or gel matrix (e.g., as described herein). Alternatively or inaddition, the first particle may be a gel bead. The first particle maybe any particle (e.g., cell or bead) described herein. For example, thesecond particle 1104A may be a cell or a cell bead comprising a polymeror gel matrix (e.g., as described herein). Alternatively or in addition,the second particle may be a gel bead. The second particle may be anyparticle (e.g., cell or bead) described herein. The first particle andthe second particle may be the same type of particle (e.g., cell orbead). The first particle and the second particle may be different typesof particles (e.g., cells or beads). For example, the first particle maybe a cell or a cell bead and the second particle may be a gel bead, orvice versa.

In a first operation 1150, the first particle 1102A and the secondparticle 1104A may be subjected to one or more conditions or one or moresets of conditions described herein to reduce a first cross-section ofthe first particle and a second cross-section of the second particle togenerate a reduced first particle 1102B and a reduced second particle1104B, respectively. In a second operation, the 1160, the reduced firstparticle 1102B and the reduced second particle 1104B may beco-partitioned in a partition 1106 (e.g., a droplet or well). Thepartition 1106 may have a cross-section smaller than a partition thatwould comprise the first particle 1102A and the second particle 1104A,prior to application of the one or more conditions or the one or moresets of conditions.

Alternatively, one or more conditions or one or more sets of conditionsmay be applied to only one of the particles (e.g., cells and/or beads),and not both. The resulting partition may still have a cross-sectionthat is smaller than a partition that would comprise the first particleand the second particle prior to application of the one or moreconditions or the one or more sets of conditions.

Alternatively, a single particle may be provided, one or more conditionsor one or more sets of conditions may be applied to the single particleand the single particle partitioned. The partition may have across-section that is smaller than a partition that would comprise thesingle particle prior to application of the one or more conditions orthe one or more sets of conditions. In some cases, a second particle maybe delivered to the partition (e.g., subsequent to provision of thefirst particle within the partition).

Alternatively, the first particle and the second particle, prior toapplication of the one or more conditions or one or more sets ofconditions, may be partitioned. The one or more conditions or one ormore sets of conditions may be applied subsequent to the partitioning toreduce cross-sections of the particles. Alternatively, the one or moreconditions or one or more sets of conditions may be applied duringpartitioning. For example, operations 1150 and 1160 may be performedsimultaneously or substantially simultaneously, whether with bothparticles or with only one of the particles.

In some cases, the one or more conditions or one or more sets ofconditions to reduce particle cross-section may be applied to the twoparticles at different times. Alternatively, one or more conditions orone or more sets of conditions to reduce particle cross-section may beapplied simultaneously.

FIG. 12A illustrates solvent-mediated results of cross-section reductionof partitions and/or particles (e.g., cells and/or beads). Panel A showspartitions 1202A which were generated without subjecting a particletherein or the partitions to one or more conditions or one or more setsof conditions described herein. Panel B shows partitions 1202B whichwere generated with subjecting a particle therein or the partitions toone or more conditions or one or more sets of conditions. Panels A and Bare shown in the same scale. As can be seen the partitions 1202B have asignificantly smaller cross-section than partitions 1202A.

FIG. 12B illustrates results of in situ cross-section reduction ofbeads. The application of one or more conditions or one or more sets ofconditions described herein to generate a cell bead (e.g., gel matrix)from a cell may concurrently or substantially concurrently reduce itscross-section, obviating a need for applying additional conditions orsets of conditions for reducing the cross-section. Discrete gel matrices1204 with reduced cross-sections are shown. In some cases, the sameconditions may also affect lysis of the cell.

A bead processed according to the methods provided herein and/or used inthe processing of a cell or component thereof may be a gel bead. Thebead may comprise a plurality of nucleic acid barcode molecules (e.g.,nucleic acid molecules each comprising a sequence comprising one or morebarcode sequences, as described herein). The bead may comprise at least10,000 nucleic acid barcode molecules attached thereto. For example, thebead may comprise at least 100,000, 1,000,000, or 10,000,000 nucleicacid barcode molecules attached thereto. Sequences of the plurality ofnucleic acid barcode molecules may be releasably attached to the bead.The sequences of the plurality of nucleic acid barcode molecules may bereleasable from the bead upon application of a stimulus. Such a stimulusmay be selected from the group consisting of a thermal stimulus, a photostimulus, and a chemical stimulus. For example, the stimulus may be areducing agent such as dithiothreitol Application of a stimulus mayresult in one or more of (i) cleavage of a linkage between the sequencesof the nucleic acid barcode molecules of the plurality of nucleic acidbarcode molecules and the bead, and (ii) degradation or dissolution ofthe bead to release sequences of the nucleic acid barcode molecules ofthe plurality of nucleic acid barcode molecules from the bead. In somecases, the nucleic acid barcode molecules may be released from the bead(e.g., upon application of a stimulus).

The plurality of nucleic acid barcode molecules attached to a beadwithin a partition (e.g., a well or droplet) comprising the cell may beuseful in a reaction involving a target molecule (e.g., target nucleicacid molecule) of the cell. For example, a nucleic acid barcode moleculemay barcode the target molecule, e.g., upon attachment by ahybridization or ligation process. In some cases, the plurality ofnucleic acid barcode molecules attached to the bead may comprise acommon barcode sequence. The plurality of nucleic acid barcode moleculesmay also comprise one or more functional sequences selected from thegroup consisting of a primer sequence, a primer annealing sequence, andan immobilization sequence. Nucleic acid barcode molecules comprising aprimer sequence may be useful in generating one or more complements orcopies of one or more nucleic acid molecules (e.g., RNA or cDNAmolecules) that may be included within the cell. For example, one ormore nucleic acid molecules included within the cell within a partition(e.g., as described herein) may be subjected to conditions suitable forperforming one or more primer extension reactions, thereby generatingextension products, where the extension reactions comprise annealing aprimer sequence of one or more of the nucleic acid barcode molecules tothe nucleic acid molecules. Access to the nucleic acid molecules of thecell may be facilitated by lysing or permeabilizing the cell (e.g., asdescribed herein). Prior to performing the extension reactions,sequences of nucleic acid barcode molecules of the plurality of nucleicacid barcode molecules may be released from the bead upon application ofa stimulus to enhance the probability of interaction between thesequences of the nucleic acid barcode molecules and the nucleic acidmolecules of the cell. In some cases, one or more amplificationreactions (e.g., PCR) may be performed to generate amplificationproducts.

FIG. 9 schematically illustrates an exemplary method for cellprocessing. Cell 902 is subjected to a first condition or set ofconditions sufficient to change a cross-section of the cell from a firstcross-section 904 to a second cross-section 910 of processed cell 908,which second cross-section 910 is less than the first cross-section 904.The first condition or set of conditions may comprise, for example, atemperature condition, a pH condition, a chemical condition, or apressure condition (e.g., as described herein). For example, the firstcondition or set of conditions may comprise a chemical species such asan organic solvent (e.g., acetone, methanol, or ethanol). Cell 902 andprocessed cell 908 may comprise target molecule 906, which may be anucleic acid molecule. Processed cell 908 may subsequently be subjectedto a second condition or set of conditions sufficient to formcross-links 914 within the cell to yield a cross-linked cell 912. Thesecond conditions or set of conditions may comprise, for example, atemperature condition, a pH condition, a chemical condition, or apressure condition (e.g., as described herein). For example, the secondcondition or set of conditions may comprise a chemical species such as across-linking agent (e.g., a photocleavable cross-linker). In somecases, the chemical species may be selected from the group consisting ofdithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS).

In an example, a cell is provided within a partition (e.g., a droplet orwell). The cell within the partition is then subjected to a firstcondition or set of conditions sufficient to change a cross-section ofthe cell from a first cross-section to a second cross-section, whichsecond cross-section is less than the first cross-section. The firstcondition or set of conditions may comprise, for example, a temperaturecondition, a pH condition, a chemical condition, or a pressure condition(e.g., as described herein). For example, the first condition or set ofconditions may comprise a chemical species such as an organic solvent(e.g., acetone, methanol, or ethanol). The cell may comprise targetmolecule that may be a nucleic acid molecule. The processed cell maysubsequently be subjected to a second condition or set of conditionssufficient to form crosslinks within the cell to yield a crosslinkedcell. The second condition or set of conditions may comprise, forexample, a temperature condition, a pH condition, a chemical condition,or a pressure condition (e.g., as described herein). For example, thesecond condition or set of conditions may comprise a chemical speciessuch as a cross-linking agent (e.g., a photocleavable cross-linker). Insome cases, the chemical species may be selected from the groupconsisting of dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyltartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS).The partition including the cell may comprise one or more other reagents(e.g., as described elsewhere herein). For example, the partition maycomprise one or more reagents for lysing or permeabilizing the cell toprovide access to the target molecule; one or more nucleic acid barcodemolecules capable of barcoding the target molecule of the cell; and oneor more other reagents.

In another example, a cell is subjected to a first condition or set ofconditions sufficient to change a cross-section of the cell from a firstcross-section to a second cross-section, which second cross-section isless than the first cross-section. The first condition or set ofconditions may comprise, for example, a temperature condition, a pHcondition, a chemical condition, or a pressure condition (e.g., asdescribed herein). For example, the first condition or set of conditionsmay comprise a chemical species such as an organic solvent (e.g.,acetone, methanol, or ethanol). The cell may comprise target moleculethat may be a nucleic acid molecule. The processed cell may subsequentlybe subjected to a second condition or set of conditions sufficient toform crosslinks within the cell to yield a crosslinked cell. The secondconditions or set of conditions may comprise, for example, a temperaturecondition, a pH condition, a chemical condition, or a pressurecondition. For example, the second condition or set of conditions maycomprise a chemical species such as a cross-linking agent (e.g., aphotocleavable cross-linker). In some cases, the chemical species may beselected from the group consisting of dithio-bis(-succinimidylpropionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycolbis(succinimidyl succinate) (EGS). The cell may be provided in anaqueous fluid. The aqueous fluid comprising the cell may then beprovided within a channel of a microfluidic device to a dropletgeneration junction and a droplet comprising the cell may be generated.The cell may then undergo further processing. The cell may beco-partitioned with one or more other reagents (e.g., as describedherein). For example, the partition (e.g., an aqueous droplet in anemulsion or a well) may comprise one or more reagents for lysing orpermeabilizing the cell to provide access to the target molecule; one ormore nucleic acid barcode molecules capable of barcoding the targetmolecule of the cell; and one or more other reagents. The cell may belysed or permeabilized to provide access to a target molecule includedtherein. The cell may be co-partitioned with a bead (e.g., a gel bead)comprising a plurality of nucleic acid barcode molecules attachedthereto. The plurality of nucleic acid barcode molecules may comprise acommon barcode sequence. The plurality of nucleic acid barcode moleculesmay also comprise one or more additional sequences as described furtherherein including, but not limited to, flow cell sequences, primersequences, sequencing primer sequences, sequences capable of coupling totarget molecule (e.g., random N-mers or poly-T sequences) uniquemolecular identifiers, and/or other sequences. Sequences of theplurality of nucleic acid barcode molecules may be releasably attachedto the bead and may be releasable from the bead upon application of astimulus (e.g., as described herein). One or more nucleic acid barcodemolecules of the plurality of nucleic acid barcode molecules or one ormore sequences thereof may participate in a reaction with the targetmolecule. For example, a nucleic acid barcode molecule or sequencethereof may hybridize to the target molecule and undergo an extensionreaction, thereby generating a complement of at least a portion of thetarget molecule. In other cases, a nucleic acid barcode molecule orsequence thereof may be coupled to the target molecule by a ligationreaction, thereby generating a barcoded target molecule. Extensionreactions may then be performed to generate a complement of the targetmolecule, or a portion thereof. In some cases, amplification reactionssuch as polymerase chain reactions (PCR) may be performed to generatecopies of the barcoded target molecule and/or its complement. Thegeneration of one or more copies or complements of a sequence ofinterest of a target molecule, or a portion thereof, (e.g., via one ormore extension and/or amplification reactions) may facilitate, forexample, detection of the sequence of interest (e.g., using sequencing).

The methods described herein may be applied to a single cell or aplurality of cells. In some cases, the plurality of cells is asuspension of cells. In some cases, at least one step of the methods ofthe present invention is performed on a single cell or a plurality ofcells outside of any partition. For instance, a method of processing aplurality of cells may comprise providing the plurality of cells withina vessel and subjecting the plurality of cells to conditions sufficientto change one or more characteristics of the cell. For example, theplurality of cells may be subjected to a first condition or set ofconditions comprising a chemical species, and a cross-section of thecells of the plurality of cells may change from a first cross-section toa second cross-section, which second cross-section is less than thefirst cross-section. The chemical species may comprise, for example, anorganic solvent such as ethanol, methanol, or acetone. The plurality ofcells may then be subjected to a second condition or set of conditionscomprising a chemical species, and crosslinks may form within each ofthe cells. The chemical species may comprise, for example, across-linking agent. The plurality of processed cells may be provided inan aqueous fluid. In some instances, the second cross-section of theplurality of cells is substantially maintained in the aqueous fluid. Theplurality of processed cells may be partitioned within a plurality ofpartitions. The partitions may be, for example, aqueous dropletsincluded in a water-in-oil emulsion. The partitions may be, for example,a plurality of wells. The plurality of fixed cells may be co-partitionedwith one or more reagents. In some cases, the plurality of fixed cellsmay be co-partitioned with one or more beads, where each bead comprisesa plurality of nucleic acid barcode molecules attached thereto. Thenucleic acid barcode molecules attached to a given bead may comprise acommon barcode sequence, and the nucleic acid barcode molecules attachedto each different bead may comprise a sequence comprising a differentcommon barcode sequence. The nucleic acid barcode molecules, or portionsthereof, may then be used in reactions with target molecules associatedwith cells of the plurality of cells.

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., biological particles, macromolecular constituents ofbiological particles, beads, reagents, etc.) into discrete compartmentsor partitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. A partition may comprise one or more other partitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles and/ormacromolecular constituents thereof. A partition may comprise one ormore gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A partition may include one ormore reagents. Alternatively, a partition may be unoccupied. Forexample, a partition may not comprise a bead. A cell bead can be abiological particle and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the biological particle andprecursors capable of being polymerized or gelled. Unique identifiers,such as barcodes, may be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead), as described elsewhere herein. Microfluidicchannel networks (e.g., on a chip) can be utilized to generatepartitions as described herein. Alternative mechanisms may also beemployed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of biological particles per partition, number of beads perpartition, etc.). For example, partition occupancy can be controlled byproviding the aqueous stream at a certain concentration and/or flow rateof particles. To generate single biological particle partitions, therelative flow rates of the immiscible fluids can be selected such that,on average, the partitions may contain less than one biological particleper partition in order to ensure that those partitions that are occupiedare primarily singly occupied. In some cases, partitions among aplurality of partitions may contain at most one biological particle(e.g., bead, DNA, cell or cellular material). In some embodiments, thevarious parameters (e.g., fluid properties, particle properties,microfluidic architectures, etc.) may be selected or adjusted such thata majority of partitions are occupied, for example, allowing for only asmall percentage of unoccupied partitions. The flows and channelarchitectures can be controlled as to ensure a given number of singlyoccupied partitions, less than a certain level of unoccupied partitionsand/or less than a certain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles,cell beads, and/or gel beads) that meet at a channel junction. Fluid maybe directed to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules anda biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule that comprises an outer shell, layer or porous matrix inwhich is entrained one or more individual biological particles or smallgroups of biological particles. The microcapsule may include otherreagents. Encapsulation of biological particles may be performed by avariety of processes. Such processes may combine an aqueous fluidcontaining the biological particles with a polymeric precursor materialthat may be capable of being formed into a gel or other solid orsemi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli can include, for example, thermalstimuli (e.g., either heating or cooling), photo-stimuli (e.g., throughphoto-curing), chemical stimuli (e.g., through crosslinking,polymerization initiation of the precursor (e.g., through addedinitiators)), mechanical stimuli, or a combination thereof.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1, may be readily used in encapsulating cells as describedherein. In particular, and with reference to FIG. 1, the aqueous fluid112 comprising (i) the biological particles 114 and (ii) the polymerprecursor material (not shown) is flowed into channel junction 110,where it is partitioned into droplets 118, 120 through the flow ofnon-aqueous fluid 116. In the case of encapsulation methods, non-aqueousfluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a virus, a cell, or a cell nucleus) ormacromolecular constituents (e.g., RNA, DNA, proteins, etc.) ofbiological particles. For example after lysing and washing the cells,inhibitory components from cell lysates can be washed away and themacromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

The polymer may be responsive to temperature. For example, the cell beadmay comprise the polymer and when heated or cooled, the characteristicsor dimensions of the cell bead may change. For example, the polymer maycomprise poly (N-isopropylacrylamide). The cell bead may comprise poly(N-isopropylacrylamide) and when heated the cell bead may decrease inone or more dimensions (e.g., a cross-sectional diameter, multiplecross-sectional diameters). A temperature sufficient for changing one ormore characteristics of the cell bead may be, for example, at leastabout 0 degrees Celsius (° C.), 1° C., 2° C., 3° C., 4° C., 5° C., 10°C., or higher. For example, the temperature may be about 4° C. In othercases, a temperature sufficient for changing one or more characteristicsof the cell may be, for example, at least about 25° C., 30° C., 35° C.,37° C., 40° C., 45° C., 50° C., or higher. For example, the temperaturemay be about 37° C.

A cell bead may include a single cell or multiple cells, or a derivativeof the single cell or multiple cells (e.g., multiple cells adheredtogether). A cell bead may include any type of cell, including withoutlimitation prokaryotic cells, eukaryotic cells, bacterial, fungal,plant, mammalian, or other animal cell types, mycoplasmas, normal tissuecells, tumor cells, a T-cell (e.g., CD4 T-cell, CD4 T-cell thatcomprises a dormant copy of human immunodeficiency virus (HIV)), a fixedcell, a cross-linked cell, a rare cell from a population of cells, orany other cell type, whether derived from single cell or multicellularorganisms. Furthermore, a cell bead may comprise a live cell, such as,for example, a cell may be capable of being cultured. Moreover, in someexamples, a cell bead may comprise a derivative of a cell, such as oneor more components of the cell (e.g., an organelle, a cell protein, acellular nucleic acid, genomic nucleic acid, messenger ribonucleic acid,a ribosome, a cellular enzyme, etc.). In some examples, a cell bead maycomprise material obtained from a biological tissue, such as, forexample, obtained from a subject. In some cases, cells, viruses ormacromolecular constituents thereof are encapsulated within a cell bead.Encapsulation can be within a polymer or gel matrix that forms astructural component of the cell bead. In some cases, a cell bead isgenerated by fixing a cell in a fixation medium or by cross-linkingelements of the cell, such as the cell membrane, the cell cytoskeleton,etc. In some cases, beads may or may not be generated withoutencapsulation within a larger cell bead.

The cell beads may also be subjected to processing as was applied tocells described elsewhere herein. In some cases, one or more physicalparameters or dimensions (e.g., diameter and volume) and/or one or moreother characteristics of the cell bead may be changed. For example, across-section of the cell bead may be changed from a first cross-sectionto a second cross-section. The first cross-section may be smaller orlarger than the second cross-section. Alternatively or in addition, oneor more other characteristics of the cell bead may be changed. Forexample, the fluidity, density, rigidity, porosity, or othercharacteristic of the cell bead or one or more components thereof may bechanged.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, all or a portion of a collection of barcoded nucleic acidmolecules can be initially associated with the microcapsule and thenreleased from the microcapsule. Release of the barcoded nucleic acidmolecules or sequences thereof can be passive (e.g., by diffusion out ofthe microcapsule). In addition or alternatively, release from themicrocapsule can be upon application of a stimulus which allows thebarcoded nucleic acid nucleic acid molecules or sequences thereof todissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules or sequences thereof to or within themicrocapsule, or both. Such stimulus can include, for example, a thermalstimulus, photo-stimulus, chemical stimulus (e.g., change in pH or useof a reducing agent(s)), a mechanical stimulus, a radiation stimulus; abiological stimulus (e.g., enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216along the channel segment 202 into junction 210. The plurality ofbiological particles 216 may be sourced from a suspension of biologicalparticles. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of biological particles 216.In some instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm,100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead may have adiameter in the range of about 40-75μm, 30-75 μm, 20-75 μm, 40-85 μm,40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor annealing to target nucleic acids, random primer, primer sequencefor messenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820, or sequences thereof, can bereleased from the bead 804 in the partition. By way of example, in thecontext of analyzing sample RNA, the poly-T segment (e.g., 812) of oneof the released nucleic acid molecule sequences (e.g., 802) canhybridize to the poly-A tail of a mRNA molecule. Reverse transcriptionmay result in a cDNA transcript of the mRNA, but which transcriptincludes each of the sequence segments 808, 810, 816 of the nucleic acidmolecule 802. Because the nucleic acid molecule sequence 802 comprisesan anchoring sequence 814, it will more likely hybridize to and primereverse transcription at the sequence end of the poly-A tail of themRNA. Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules may include a common barcode sequence segment810. However, the transcripts made from the different mRNA moleculeswithin a given partition may vary at the unique molecular identifyingsequence 812 segment (e.g., UMI segment). Beneficially, even followingany subsequent generation of one or more copies or complements of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the biological particle (e.g., cell). As noted above, oneor more copies or complements of the transcripts can be generated,cleaned up and sequenced to identify the sequence of the cDNA transcriptof the mRNA, as well as to sequence the barcode segment and the UMIsegment. While a poly-T primer sequence is described, other targeted orrandom priming sequences may also be used in priming the reversetranscription reaction. Likewise, although described as releasing thebarcoded oligonucleotides or sequences thereof into the partition, insome cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. For example, abead may comprise oligonucleotides comprising releasable sequencescomprising barcodes. A bead injected or otherwise introduced into apartition may comprise activatable barcodes. A bead injected orotherwise introduced into a partition may be degradable, disruptable, ordissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide, or sequencethereof) may result in release of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides orsequences thereof) are released within the droplet when the appropriatestimulus is applied. The free species (e.g., oligonucleotides, nucleicacid molecules, or sequences thereof) may interact with other reagentscontained in the partition. For example, a polyacrylamide beadcomprising cystamine and linked, via a disulfide bond, to a barcodesequence, may be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent can breakthe various disulfide bonds, resulting in bead degradation and releaseof the barcode sequence into the aqueous, inner environment of thedroplet. In another example, heating of a droplet comprising abead-bound barcode sequence in basic solution may also result in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) or sequences thereof are present in the partition at apre-defined concentration. Such pre-defined concentration may beselected to facilitate certain reactions for generating a sequencinglibrary, e.g., primer extension reactions or amplification reactions,within the partition. In some cases, the pre-defined concentration ofthe primer can be limited by the process of producing nucleic acidmolecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the sequence of the oligonucleotide, e.g., a barcode containingsequence, from the bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcode sequences that are releasably, cleavably orreversibly attached to the beads described herein (e.g., as componentsof sequences of nucleic acid barcode molecules or oligonucleotides)include barcode sequences that are released or releasable throughcleavage of a linkage between the barcode molecule and the bead, or thatare released through degradation of the underlying bead itself, allowingthe barcodes to be accessed or accessible by other reagents, or both.

The barcode sequences that are releasable as described herein maysometimes be referred to as being activatable, in that they areavailable for reaction once released. Thus, for example, an activatablebarcode may be activated by releasing the barcode sequence from a bead(or other suitable type of partition described herein). Otheractivatable configurations are also envisioned in the context of thedescribed methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid extension and/or amplification reaction (e.g., primers,polymerases, dNTPs, co-factors (e.g., ionic co-factors), buffers)including those described herein, reagents for enzymatic reactions(e.g., enzymes, co-factors, substrates, buffers), reagents for nucleicacid modification reactions such as polymerization, ligation, ordigestion, and/or reagents for template preparation (e.g., tagmentation)for one or more sequencing platforms (e.g., Nextera® for Illumina®).Such species may include one or more enzymes described herein, includingwithout limitation, polymerase, reverse transcriptase, restrictionenzymes (e.g., endonuclease), transposase, ligase, proteinase K, DNAse,etc. Such species may include one or more reagents described elsewhereherein (e.g., lysis agents, inhibitors, inactivating agents, chelatingagents, stimulus). Trapping of such species may be controlled by thepolymer network density generated during polymerization of precursors,control of ionic charge within the gel bead (e.g., via ionic specieslinked to polymerized species), or by the release of other species.Encapsulated species may be released from a bead upon bead degradationand/or by application of a stimulus capable of releasing the speciesfrom the bead. Alternatively or in addition, species may be partitionedin a partition (e.g., droplet) during or subsequent to partitionformation. Such species may include, without limitation, theabovementioned species that may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc.) or component thereof from the bead when the appropriate stimulusis applied to the bead as compared to a bead that does not degrade. Forexample, for a species bound to an inner surface of a porous bead or inthe case of an encapsulated species, the species may have greatermobility and accessibility to other species in solution upon degradationof the bead. In some cases, a species may also be attached to adegradable bead via a degradable linker (e.g., disulfide linker). Thedegradable linker may respond to the same stimuli as the degradable beador the two degradable species may respond to different stimuli. Forexample, a barcode sequence may be attached, via a disulfide bond, to apolyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releaseof the molecule tag molecules or components thereof (e.g., sequencesthereof, such as sequences comprising barcode sequences) from the bead,the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) or components thereof are present in the partition at apre-defined concentration. Such pre-defined concentration may beselected to facilitate certain reactions for generating a sequencinglibrary, e.g., extension reactions or amplification reactions, withinthe partition. In some cases, the pre-defined concentration of theprimer can be limited by the process of producing oligonucleotidebearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100pL, 50 pL, 20pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, 100pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles' contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with thebiological particles described above, other reagents can also beco-partitioned with the biological particles, including, for example,DNase and RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated biological particles, thebiological particles may be exposed to an appropriate stimulus torelease the biological particles or their contents from a co-partitionedmicrocapsule. For example, in some cases, a chemical stimulus may beco-partitioned along with an encapsulated biological particle to allowfor the degradation of the microcapsule and release of the cell or itscontents into the larger partition. In some cases, this stimulus may bethe same as the stimulus described elsewhere herein for release ofnucleic acid molecules (e.g., oligonucleotides) or sequences thereoffrom their respective microcapsule (e.g., bead). In alternative aspects,this may be a different and non-overlapping stimulus, in order to allowan encapsulated biological particle to be released into a partition at adifferent time from the release of nucleic acid molecules or sequencesthereof into the same partition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to generate copies orcomplements of the biological particle's nucleic acid fragments and toattach the barcode molecular tags to the copies or complements. Otherenzymes may be co-partitioned, including without limitation, polymerase,transposase, ligase, proteinase K, DNAse, etc. Additional reagents mayalso include reverse transcriptase enzymes, including enzymes withterminal transferase activity, primers and oligonucleotides, and switcholigonucleotides (also referred to herein as “switch oligos” or“template switching oligonucleotides”) which can be used for templateswitching. In some cases, template switching can be used to increase thelength of a cDNA. In some cases, template switching can be used toappend a predefined nucleic acid sequence to the cDNA. In an example oftemplate switching, cDNA can be generated from reverse transcription ofa template, e.g., cellular mRNA, where a reverse transcriptase withterminal transferase activity can add additional nucleotides, e.g.,polyC, to the cDNA in a template independent manner. Switch oligos caninclude sequences complementary to the additional nucleotides, e.g.,polyG. The additional nucleotides (e.g., polyC) on the cDNA canhybridize to the additional nucleotides (e.g., polyG) on the switcholigo, whereby the switch oligo can be used by the reverse transcriptaseas template to further extend the cDNA. Template switchingoligonucleotides may comprise a hybridization region and a templateregion. The hybridization region can comprise any sequence capable ofhybridizing to the target. In some cases, as previously described, thehybridization region comprises a series of G bases to complement theoverhanging C bases at the 3′ end of a cDNA molecule. The series of Gbases may comprise 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G basesor more than 5 G bases. The template sequence can comprise any sequenceto be incorporated into the cDNA. In some cases, the template regioncomprises at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. Switch oligos may comprise deoxyribonucleicacids; ribonucleic acids; modified nucleic acids including2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or anycombination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particles, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2). In someaspects, the unique identifiers are provided in the form of nucleic acidmolecules (e.g., oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal primer sequences (e.g., universalamplification primer sequences) for generating copies and/or complementsof (e.g., amplifying) the genomic DNA from the individual biologicalparticles within the partitions while attaching the associated barcodesequences, sequencing primers or primer recognition sites, hybridizationor probing sequences, e.g., for identification of presence of thesequences or for pulling down barcoded nucleic acids, or any of a numberof other potential functional sequences. Other mechanisms ofco-partitioning oligonucleotides may also be employed, including, e.g.,coalescence of two or more droplets, where one droplet containsoligonucleotides, or microdispensing of oligonucleotides intopartitions, e.g., droplets within microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) attached to the beads, whereall of the nucleic acid molecules attached to a particular bead willinclude the same nucleic acid barcode sequence, but where a large numberof diverse barcode sequences are represented across the population ofbeads used. In some embodiments, hydrogel beads, e.g., comprisingpolyacrylamide polymer matrices, are used as a solid support anddelivery vehicle for the nucleic acid molecules into the partitions, asthey are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules orsequences thereof upon exposure to a particular stimulus, as describedelsewhere herein. In some cases, the population of beads provides adiverse barcode sequence library that includes at least about 1,000different barcode sequences, at least about 5,000 different barcodesequences, at least about 10,000 different barcode sequences, at leastabout 50,000 different barcode sequences, at least about 100,000different barcode sequences, at least about 1,000,000 different barcodesequences, at least about 5,000,000 different barcode sequences, or atleast about 10,000,000 different barcode sequences, or more.Additionally, each bead can be provided with large numbers of nucleicacid (e.g., oligonucleotide) molecules attached. In particular, thenumber of molecules of nucleic acid molecules including the barcodesequence on an individual bead can be at least about 1,000 nucleic acidmolecules, at least about 5,000 nucleic acid molecules, at least about10,000 nucleic acid molecules, at least about 50,000 nucleic acidmolecules, at least about 100,000 nucleic acid molecules, at least about500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules,at least about 5,000,000 nucleic acid molecules, at least about10,000,000 nucleic acid molecules, at least about 50,000,000 nucleicacid molecules, at least about 100,000,000 nucleic acid molecules, atleast about 250,000,000 nucleic acid molecules and in some cases atleast about 1 billion nucleic acid molecules, or more. Nucleic acidmolecules of a given bead can include identical (or common) barcodesequences, different barcode sequences, or a combination of both.Nucleic acid molecules of a given bead can include multiple sets ofnucleic acid molecules. Nucleic acid molecules of a given set caninclude identical barcode sequences. The identical barcode sequences canbe different from barcode sequences of nucleic acid molecules of anotherset.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) or sequences thereofare releasable from the beads upon the application of a particularstimulus to the beads. In some cases, the stimulus may be aphoto-stimulus, e.g., through cleavage of a photo-labile linkage thatreleases the sequences of the nucleic acid molecules. In other cases, athermal stimulus may be used, where elevation of the temperature of thebeads environment will result in cleavage of a linkage or other releaseof the sequences of the nucleic acid molecules form the beads. In stillother cases, a chemical stimulus can be used that cleaves a linkage ofthe nucleic acid molecules to the beads, or otherwise results in releaseof the sequences of the nucleic acid molecules from the beads. In onecase, such compositions include the polyacrylamide matrices describedabove for encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules or sequences thereofthrough exposure to a reducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan \; \alpha}\frac{w}{h_{0}}}} \right)\mspace{11mu} \frac{h_{0}}{\sqrt{\tan \; \alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°,40°, 45°, 50°, 55°,60°, 65°, 70°, 75°, 80°, 85°or higher.

In some instances, the expansion angle can be at most about 89°, 88°,87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°,45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°,1°, 0.1°, 0.01°, or less. In some instances, the width, w, can bebetween a range of from about 100 micrometers (μm) to about 500 μm. Insome instances, the width, w, can be between a range of from about 10 μmto about 200 μm. Alternatively, the width can be less than about 10 μm.Alternatively, the width can be greater than about 500 μm. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.04 microliters (μL)/minute (min) and about 40μL/min. In some instances, the flow rate of the aqueous fluid 408entering the junction 406 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 408 entering the junction 406 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min , 120 μL/min, 130 μL/min , 140 μL/min , 150 μL/min, or greater.At lower flow rates, such as flow rates of about less than or equal to10 microliters/minute, the droplet radius may not be dependent on theflow rate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, α (not shown in FIG. 6)at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,βh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min , 120 μL/min , 130 μL/min , 140 μL/min , 150 μL/min, or greater.At lower flow rates, such as flow rates of about less than or equal to10 microliters/minute, the droplet radius may not be dependent on theflow rate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, zlh, beingabrupt at the junction 706 (e.g., a step increase), the heightdifference may increase gradually (e.g., from about 0 μm to a maximumheight difference). Alternatively, the height difference may decreasegradually (e.g., taper) from a maximum height difference. A gradualincrease or decrease in height difference, as used herein, may refer toa continuous incremental increase or decrease in height difference,wherein an angle between any one differential segment of a heightprofile and an immediately adjacent differential segment of the heightprofile is greater than 90°. For example, at the junction 706, a bottomwall of the channel and a bottom wall of the reservoir can meet at anangle greater than 90°. Alternatively or in addition, a top wall (e.g.,ceiling) of the channel and a top wall (e.g., ceiling) of the reservoircan meet an angle greater than 90°. A gradual increase or decrease maybe linear or non-linear (e.g., exponential, sinusoidal, etc.).Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly. While FIGS. 7A and 7Billustrate the expanding reservoir cross-section height as linear (e.g.,constant expansion angle, β), the cross-section height may expandnon-linearly. For example, the reservoir may be defined at leastpartially by a dome-like (e.g., hemispherical) shape having variableexpansion angles. The cross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of extension and/oramplification products, purification (e.g., via solid phase reversibleimmobilization (SPRI)), and further processing (e.g., shearing, ligationof functional sequences, and subsequent generation of copies and/orcomplements of target sequences (e.g., amplification, such as via PCR)).These operations may occur in bulk (e.g., outside the partition). In thecase where a partition is a droplet in an emulsion, the emulsion can bebroken and the contents of the droplet pooled for additional operations.Additional reagents that may be co-partitioned along with the barcodebearing bead may include oligonucleotides to block ribosomal RNA (rRNA)and nucleases to digest genomic DNA from cells. Alternatively, rRNAremoval agents may be applied during additional processing operations.The configuration of the constructs generated by such a method can helpminimize (or avoid) sequencing of the poly-T sequence during sequencingand/or sequence the 5′ end of a polynucleotide sequence. Extensionproducts, for example, first extension products and/or second extensionproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 10 shows a computer system1001 that is programmed or otherwise configured to, for example, (i)control a microfluidics system (e.g., fluid flow), (ii) sort occupieddroplets from unoccupied droplets, (iii) polymerize droplets, (iv)perform sequencing applications, or (v) generate and maintain a libraryof nucleic acid molecules. The computer system 1001 can regulate variousaspects of the present disclosure, such as, for example, fluid flowrates in one or more channels in a microfluidic structure,polymerization application units, etc. The computer system 1001 can bean electronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 1001 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1005, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1001 also includes memory or memorylocation 1010 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1015 (e.g., hard disk), communicationinterface 1020 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1025, such as cache, othermemory, data storage and/or electronic display adapters. The memory1010, storage unit 1015, interface 1020 and peripheral devices 1025 arein communication with the CPU 1005 through a communication bus (solidlines), such as a motherboard. The storage unit 1015 can be a datastorage unit (or data repository) for storing data. The computer system1001 can be operatively coupled to a computer network (“network”) 1030with the aid of the communication interface 1020. The network 1030 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1030 insome cases is a telecommunication and/or data network. The network 1030can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1030, in some cases withthe aid of the computer system 1001, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1001 tobehave as a client or a server.

The CPU 1005 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1010. The instructionscan be directed to the CPU 1005, which can subsequently program orotherwise configure the CPU 1005 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1005 can includefetch, decode, execute, and writeback.

The CPU 1005 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1001 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1015 can store files, such as drivers, libraries andsaved programs. The storage unit 1015 can store user data, e.g., userpreferences and user programs. The computer system 1001 in some casescan include one or more additional data storage units that are externalto the computer system 1001, such as located on a remote server that isin communication with the computer system 1001 through an intranet orthe Internet.

The computer system 1001 can communicate with one or more remotecomputer systems through the network 1030. For instance, the computersystem 1001 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1001 via the network 1030.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1001, such as, for example, on thememory 1010 or electronic storage unit 1015. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1005. In some cases, thecode can be retrieved from the storage unit 1015 and stored on thememory 1010 for ready access by the processor 1005. In some situations,the electronic storage unit 1015 can be precluded, andmachine-executable instructions are stored on memory 1010.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1001, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1001 can include or be in communication with anelectronic display 1035 that comprises a user interface (UI) 1040 forproviding, for example, results of sequencing analysis, etc. Examples ofUIs include, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1005. Thealgorithm can, for example, perform sequencing, etc.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is: 1.-82. (canceled)
 83. A method of processing a cell,comprising: (a) subjecting said cell to conditions sufficient to: (i)change a cross-section of said cell from a first cross-section to asecond cross-section, which second cross-section is less than said firstcross-section, and (ii) form crosslinks within said cell having saidsecond cross-section; and (b) providing said cell having said secondcross-section in an aqueous fluid.
 84. The method of claim 83, whereinsaid crosslinks are formed upon cross-linking one or more cross-linkablemolecules within said cell.
 85. The method of claim 84, wherein said oneor more cross-linkable molecules are one or more polymers.
 86. Themethod of claim 83, wherein said crosslinks are formed upon polymerizinga plurality of monomers within said cell.
 87. The method of claim 83,wherein said cross-section of said cell is changed from said firstcross-section to said second cross-section concurrently with formationof said crosslinks within said cell.
 88. The method of claim 83, whereinsaid crosslinks are formed subsequent to changing said cross-sectionfrom said first cross-section to said second cross-section.
 89. Themethod of claim 83, wherein said second cross-section is substantiallymaintained in said aqueous fluid.
 90. The method of claim 83, whereinsaid aqueous fluid is in a droplet as part of an emulsion.
 91. Themethod of claim 90, wherein the volume of said droplet is less than10,000 pL.
 92. The method of claim 83, wherein (a) comprises bringingsaid cell in contact with a first chemical species and a second chemicalspecies, wherein (i) comprises using said first chemical species tochange said cross-section from said first-cross-section to said secondcross-section, and wherein (ii) comprises using said second chemicalspecies to form said crosslinks within said cell.
 93. The method ofclaim 92, wherein said first or second chemical species is selected fromthe group consisting of disuccinimidyl suberate (DSS),dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA),dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate(DST), and ethylene glycol bis(succinimidyl succinate) (EGS).
 94. Themethod of claim 92, wherein said first or said second chemical speciesis selected from the group consisting of an organic solvent and across-linking agent.
 95. The method of claim 94, wherein said organicsolvent is acetone or an alcohol.
 96. The method of claim 94, whereinsaid cross-linking agent is selected from the group consisting of aphotocleavable crosslinker and an aldehyde.
 97. The method of claim 83,further comprising providing said cell in an aqueous reaction mixture,wherein said cell comprises a target molecule, and performing one ormore reactions using said target molecule.
 98. The method of claim 97,further comprising co-partitioning said cell in a partition andperforming said one or more reactions in said partition.
 99. The methodof claim 98, wherein said partition is a droplet or a well.
 100. Themethod of claim 98, wherein said target molecule is a nucleic acidmolecule and wherein said partition further comprises a plurality ofnucleic acid barcode molecules, wherein each nucleic acid barcodemolecule of the plurality of nucleic acid barcode molecules comprises asequence comprising a common barcode sequence.
 101. The method of claim100, wherein said plurality of nucleic acid barcode molecules areattached to a bead.
 102. The method of claim 101, wherein said pluralityof nucleic acid barcode molecules are releasably attached to a bead, andthe method further comprises releasing said sequences of said pluralityof nucleic acid barcode molecules from said bead within said partition.103. The method of claim 101, wherein said bead is a gel bead.
 104. Themethod of claim 103, wherein said gel bead is degradable uponapplication of a stimulus.
 105. The method of claim 104, wherein saidstimulus is a chemical stimulus.
 106. The method of claim 83, whereinsaid cross-section is a diameter or a volume of said cell.
 107. Themethod of claim 83, wherein said second cross-section of said cell isreduced by at least 5% compared to said first cross-section.
 108. Themethod of claim 83, wherein said change from said first cross-section ofsaid cell to said second cross-section is irreversible.
 109. The methodof claim 83, wherein said change from said first cross-section of saidcell to said second cross-section is reversible upon application of astimulus.
 110. The method of claim 109, wherein said stimulus isselected from the group consisting of a thermal stimulus, a photostimulus, and a chemical stimulus.
 111. The method of claim 109, furthercomprising applying said stimulus, wherein application of said stimulusreverses said change from said first cross-section to said secondcross-section by at least 75%.